The present invention relates to oligosaccharides of formula: 
or mixtures thereof, to diastereoisomers thereof, to a process for their preparation and to pharmaceutical compositions containing them.
Disaccharide sulfates containing a 1,6-anhydro structure at the reducing end have been described by H. P. Wessel, J. Carbohydrate Chemistry, 11(8), 1039-1052 (1992); no pharmacological activity is mentioned for these products.
Trisaccharide sulfates comprising a 1,6-anhydro unit have also been described in patent EP 84999 and by Y. Ichikawa et al., Carbohyd. Res, 141, 273-282 (1985) as intermediates for preparing higher oligosaccharides. These trisaccharides have low anti-factor Xa activity.
In formula (I) n is 0 or an integer from 1 to 25; R1, R3, R4 and R5, which may be identical or different, represent a hydrogen atom or an SO3M radical; R2 and R6, which may be identical or different, represent a hydrogen atom or a radical selected from SO3M and COCH3; and M is sodium, calcium, magnesium or potassium.
These oligosaccharides thus comprise an even number of saccharide units.
In formula (I), R4 is preferably a hydrogen atom.
Preferably, n is 0 or an integer from 1 to 10; more preferably 0 or an integer from 1 to 6; even more preferably an integer from 1 to 6.
The oligosaccharides of formula (I) can be prepared by the action of an alkali metal or quaternary ammonium hydroxide on oligosaccharides of formula: 
in which n is 0 or an integer from 1 to 25; R1, R3, R4 and R5, which may be identical or different, represent a hydrogen atom or an SO3M radical; R2 and R6, which may be identical or different, represent a hydrogen atom or a radical selected from SO3M and COCH3 and M is sodium, calcium, magnesium or potassium, or a mixture thereof.
This reaction is carried out in aqueous medium, at a temperature of from 40 to 80xc2x0 C., at a pH of from 10 to 13.
As alkali metal hydroxides which can be used, mention may be made of sodium hydroxide, potassium hydroxide, lithium hydroxide and cesium hydroxide.
As a quaternary ammonium hydroxide which may be used, mention may be made of tetrabutylammonium hydroxide.
The amount of alkali metal or quaternary ammonium hydroxide must be sufficient for the pH of the reaction medium to remain stable throughout the reaction. It is thus necessary to add the alkali metal or quaternary ammonium hydroxide continuously throughout the reaction.
Preferably, the alkali metal or quaternary ammonium hydroxide is in the form of an aqueous 1 to 5% solution.
Preferably, the reaction is carried out at a temperature of from 60 to 70xc2x0 C.
Advantageously, the reaction pH is from 11 to 12.5.
The reaction is stopped by acidifying the reaction medium, for example by addition of acidic resin such as Amberlite IR120(copyright) resin (Fluka).
The oligosaccharides of formula (I) can be eventually purified by gel permeation chromatography with polyacrylamide-agarose type gel such as Ultrogel ACA202 (R) (Biosepra) as described hereinafter for the intermediate oligosaccharides of formula (II).
The oligosaccharides of formula (I) for which n is 0 or 1 can be also eventually purified on an alumina column with a water-ethanol mixture as eluant.
The intermediate oligosaccharides of formula (II) and mixtures thereof can be obtained by chromatographic separation on gel of a mixture of oligosaccharides (III) obtained by enzymatic depolymerization of heparin or basic depolymerization of the benzyl ester of heparin or of a benzyl ester of semi-synthetic heparin.
This chromatography is carried out on columns filled with gel of polyacrylamide-agarose type such as the gel sold under the brand name Ultrogel ACA202(copyright) (Biosepra). Preferably, an array of polyacrylamide agarose gel columns is used. The number of columns used is adapted as a function of the volume, of the gel and of the oligosaccharides to be separated. The mixture is eluted with a solution containing a phosphate buffer and sodium chloride. Preferably, the phosphate buffer is a solution containing 0.02 mol/l of NaH2PO4/Na2HPO4 (pH 7) containing 0.1 mol/l of sodium chloride. The detection of the various fractions is carried out by UV spectrometry (254 nm) and ionic spectrometry (IBF). The fractions thus obtained can then be optionally purified, for example by SAX (strong anion exchange) chromatography according to the methods known to those skilled in the art and in particular according to the methods described by K. G. Rice and R. J. Linhardt, Carbohydrate Research 190, 219-233 (1989), A. Larnkjaer, S. H. Hansen and P. B. Ostergaard, Carbohydrate Research, 266, 37-52 (1995) and in patent WO 90/01501 (Example 2). The fractions are then freeze-dried, after which they are desalified on a column filled with gel such as a column of Sephadex G10(copyright) gel (Pharmacia Biochemicals).
When the purification is not carried out by SAX chromatography, the lyophilizates can be optionally purified by simple or fractional precipitation according to the methods known to those skilled in the art and in particular according to the method described in patent FR 2 548 672. In general, the process is performed according to the following procedure:
The lyophilized fraction to be purified is dissolved at 25xc2x0 C. in about ten volumes of distilled water. On adding methanol or ethanol, the desired oligosaccharide is precipitated, while monitoring its enrichment by HPLC chromatography (high performance liquid chromatography). The addition of methanol or ethanol is determined as a function of the desired yield and purity of the said oligosaccharide. Similarly, this operation can be carried out in several successive steps starting with the initial solution of lyophilizate. For this, more of the insolubilizing agent (methanol or ethanol) is added portionwise and the precipitate obtained after each addition is isolated. The precipitates thus prepared are analyzed by HPLC chromatography. Depending on the desired yield and purity, the suitable-fractions of precipitate are combined.
For the intermediates of formula (II) for which n=0, 1 or 2, it is preferable to start with a mixture (III) obtained by enzymatic depolymerization of heparin.
This depolymerization is carried out by means of heparinase I (EC 4.2.2.7), in a pH 7 phosphate buffer solution, in the presence of sodium chloride and BSA (bovine serum albumin), at a temperature of between 10 and 18xc2x0 C., and preferably 15xc2x0 C., for 8 to 10 days, and preferably 9 days. The depolymerization is stopped, for example, by heating the reaction medium at 100xc2x0 C. for 2 minutes, and the mixture is recovered by lyophilization. It is preferable to use 7 IU of heparinase I per 25 g of heparin. The phosphate buffer solution generally comprises 0.05 mol/l of NaH2PO4/Na2HPO4 (pH 7) in the presence of 0.1 mol/l of sodium chloride. The BSA concentration is generally 2%.
For the intermediates of formula (II) for which n=0, 1, 2, 3 or 4, it is,preferable to start with a mixture (III) obtained by depolymerizing a benzyl ester of heparin.
The benzyl ester of heparin can be prepared according to the methods described in patents U.S. Pat. No. 5 389 618, EP 40 144 and FR 2 548 672. The degree of esterification will preferably be between 50 and 100%. More preferably, it will be between 70 and 90%.
The depolymerization is carried out in aqueous medium, by means of an alkali metal hydroxide (for example lithium hydroxide, sodium hydroxide, potassium hydroxide or cesium hydroxide) or of a quaternary ammonium hydroxide (for example tetrabutylammonium hydroxide), preferably at a molarity of between 0.1 and 0.2 mol/l, at a temperature of between 40 and 80xc2x0 C., for 5 to 120 minutes. In one preferred mode, the process is performed for 5 to 15 minutes, at a temperature of between 60 and 70xc2x0 C., with a 0.15 mol/l sodium hydroxide solution. The depolymerization reaction is stopped by neutralization by addition of an acid such as acetic acid. After addition of 10% by weight per volume of sodium acetate, the oligosaccharide mixture is precipitated by adding methanol, preferably 2 volumes per 1 volume of reaction medium, and filtered.
According to one preferred aspect of the invention, the oligosaccharide mixture obtained after chemical depolymerization, in the form of an aqueous solution, is enriched by ultrafiltration through membranes with a suitable nominal cutoff threshold (of the Pellicon type made with regenerated cellullose, sold by Millipore); the type of membrane being adapted as a function of the type of enriched oligosaccharides to be recovered. For the oligosaccharides (II) for which n=0, a membrane with a nominal cutoff threshold of 1 kDa will be used, for the oligosaccharides (II) for which n=1, a 1 kDa or 3 kDa membrane will be used, for the oligosaccharides (II) for which n=2, a 3 kDa membrane will be used, and for the oligosaccharides (II) for which n=3 or 4, a 5 kDa. membrane will be used. During this operation, the permeate is recovered and the retentate is discarded. Thus, the fraction of enriched product can represent from 50 to 10% of the initial oligosaccharide mixture while at the same time conserving at least 80% of the desired oligosaccharide.
For the intermediates of formula (II) for which n=0 to 25, it is preferable to start with a mixture (III) obtained by depolymerizing a benzyl ester or semi-synthetic polysaccharide sulfate. The benzyl ester of semi-synthetic polysaccharide sulfate is prepared from semi-synthetic polysaccharide sulfates"" obtained from polysaccharide K5 and according to the. methods described in the patents WO 94/29352 and WO 96/14425. The esterification, depolymerization and recovery conditions are the same as those described above for the benzyl ester of heparin.
In all the preceding processes, the initial heparin can be of porcine, ovine, caprine or bovine origin and can be obtained from the mucus, lungs or hides of the animals. Preferably, a heparin from porcine or ovine mucus or from bovine lung is used, and even more preferably from porcine mucus.
The oligosaccharides of formula (I) have anti-inflammatory properties and can thus be used for preventing or treating diseases associated with an inflammatory process involving the production of cytotoxic substances such as nitrogen monoxide (NO) whose inducible form is released in particular by neutrophils or macrophages when the latter migrate and are activated in a tissue. The activation, migration, adhesion and infiltration of neutrophils takes place in ischemic tissue regions following an occlusion or spasm of an artery vascularizing this tissue. These ischemias can arise either in the brain (cerebrovascular accident) or in the myocardium (myocardial infarction) or in the lower limbs (so-called peripheral ischemias). The oligosaccharides of formula (I) can thus be used for the prevention and/or treatment of neurodegenerative diseases for which such inflammation plays a deleterious role which can lead to death, among which mention may be made of cerebral ischemias, cardiac ischemias (myocardial infarction), peripheral ischemias, traumas of the central nervous system, in particular, cranial, spinal and craniospinal traumas, multiple sclerosis, neuropathic pain and peripheral neuropathies, motor neuron diseases including amyotrophic lateral sclerosis, neuro-AIDS, Alzheimer""s disease, Parkinson""s disease and Huntington""s chorea and certain forms of osteoarthritis, in particular of articular localization.
The anti-inflammatory activity of these products is demonstrated in vivo in the test of production of NOx (nitrite and nitrate) induced by a lipopolysaccharide (LPS) obtained from E. coli according to the protocol described by M. Yamashita et al., Eur. J. Pharmacol, 338, 2, 151-158 (1997) or J. E. Shellito et al., Am. J. Respir. Cell Mol. Biol., 13, 1, 45-53 (1995).
0.5 mg/kg of the oligosaccharide are injected into male CD1 mice (Charles River, 25-35 g) at TO via intravenous bolus, and 1 or 2 mg/kg of the oligosaccharide are injected subcutaneously at T+15 minutes. At T+30 minutes, 100 mg/kg of lipopolysaccharide (LPS) obtained from E. coli. (Sigma L3129, serotype 0127:B8) are injected. A further 1 or 2 mg/kg of the oligosaccharide are injected subcutaneously at T+3 hours. At T+5 hours 30 minutes, a blood sample is collected by ocular puncture and the concentrations of NOx (nitrite and nitrate) in the plasma are determined by the Griess colorimetric method after reduction of the nitrate to nitrite with nitrate reductase in the following way: 12 xcexcl of the plasma sample are mixed with 88 xcexcl of deionized water and incubated in the dark for 1 hour at room temperature with 40 xcexcl of phosphate buffer (0.31 M, pH 7.5), 20 xcexcl of xcex2-NADPH (reduced nicotinamide adenine dinucleotide phosphate) (0.86 mM), 20 xcexcl of FDA (flavin adenine dinucleotide) (0.11 mM) and 20 xcexcl of nitrate reductase (2 U/ml) (Boehringer Mannheim). 10 xcexcl of ZnSO4 (1M) are added to precipitate the proteins and, after mixing, the samples are centrifuged at 20,000xc3x97g for 5 minutes. Finally, 50 xcexcl of the supernatant are incubated for 10 minutes at room temperature with 100 xcexcl of the Griess reagent (1% sulfanilamide in a phosphoric acid/0.1% naphthylethylenediamine mixture in deionized water (V/V)). The optical densities are read at 540 nm with a microplate spectrophotometer; each point being determined twice. KNO3 and NaNO2 are used as standards for the calorimetric method.
In this test, the oligosaccharides according to the invention inhibit the formation of NOx by more than 50%.
Among the oligosaccharides of formula (I) which are preferred, mention may be made in particular of the oligosaccharides for which:
n is equal to 0, R1 and R6 represent an SO3Na radical and M is sodium, and the mixture of its diastereoisomers;
n is equal to 1, R1, R2, R3, R5 and R6 represent an SO3Na radical, R4 represents a hydrogen atom and M is sodium, and the mixture of its diastereoisomers;
n is equal to 2, R1, R2, R3, R5 and R6 represent an SO3Na radical, R4 represents a hydrogen atom and M is sodium, and the mixture of its diastereoisomers;
n is equal to 2, R1, R2, R3 and R6 represent an SO3Na radical, R5 represents a hydrogen atom or an SO3Na radical, R4 represents a hydrogen atom and M is sodium, and the mixture of its diastereoisomers (1,6-anhydro xcex94Is-Is-IIs derivative).